DNA encoding chimeric IgG Fc receptor

ABSTRACT

The present invention relates, in general, to methods of stimulating phagocytosis and thereby combating infection and/or modulating immune complex disease, in particular, to methods of modulating the number and type of Fc receptors present on cells that normally possess such receptors, including monocytes and macrophages, as well as on cells that normally do not possess Fc receptors, such as fibroblasts, and to compounds and compositions suitable for use in such methods.

This is a divisional of application Ser. No. 08/129,391, filed Sep. 30,1993, now abandoned.

TECHNICAL FIELD

The present invention relates, in general, to methods of stimulatingphagocytosis and thereby combating infection and/or modulating immunecomplex disease, in particular, to methods of modulating the number andtype of Fc receptors present on cells that normally possess suchreceptors, including monocytes and macrophages, as well as on cells thatnormally do not possess Fc receptors, such as fibroblasts, and tocompounds and compositions suitable for use in such methods.

BACKGROUND

Mononuclear phagocytes (blood monocytes and tissue macrophages) havecell surface receptors for the Fc domain of IgG antibody. Thesereceptors (FCγR) mediate humoral immune effector functions includingphagocytosis, clearance of immune complexes and antibody-dependent cellcytotoxicity. Three classes of Fcγ receptors have been identified onhuman cells and characterized on the basis of size, primary structure,binding affinity for IgG subclasses, and recognition by monoclonalantibodies: FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). FcγRI is ahigh affinity receptor, expressed on resting mononuclear phagocytes andstimulated neutrophils. FcγRII and FcγRIII are low affinity receptorsfound on a range of hematopoietic cells, including monocytes andmacrophages. Macrophages express all three receptor classes whilemonocytes express primarily FcγRI and FcγRII.

All three classes of human Fcγ receptors have been isolated and cloned(Allen and Seed, Science 243:378 (1989); Hibbs et al, Proc. Natl. Acad.Sci. USA 85:2240 (1988); and J. Exp. Med. 166:1668 (1987)). At least twogenes code for the FcγRI class of receptors (van de Winkle et al, FASEBJ. 5:A964 (1991)), three genes code for the FcγRII class (designatedFcγRIIA, FcγRIIB and FcγRIIC) (Brooks et al, J. Exp. Med. 170:369(1989); Stuart et al, EMBO J. 8:3657 (1989); Qui et al, Science 248:732(1990)) and two genes code for the FcγRIII receptor class (Simmons andSeed, Nature 333:568 (1988)).

Macrophage Fcγ receptors participate in the clearance of IgG-coatedparticulate and soluble antigens, including IgG-coated microorganisms,and thereby remove potentially dangerous foreign organisms. Due to theirimportance in host defense, functional integrity of Fcγ receptors hasbeen studied in connection with various disease states, includingautoimmune disorders (Frank et al, Ann. Intern. Med. 98:206 (1983);Kimberley and Ralph, Am. J. Med. 74:481 (1983)) and end-stage renaldisease (Ruiz et al, N. Engl. J. Med. 322:717 (1990)). Macrophage Fcγreceptor function has been found to be decreased in patients withcertain HLA haplotypes and in patients with the immune disorderssystemic lupus erythematosus, Sjogren's syndrome and dermatitisherpetiformis (this observation was attributed to occupation of thesereceptors on the macrophages by immune complexes). In end-stage renaldisease, macrophage Fcγ receptor function has been found to be impairedand this impairment is believed to contribute to the observedimmunodepression among such patients.

Various diseases, non-bacterial in origin, are associated with a highincidence of complications due to infection. Examples of such diseasesinclude the above-noted end-stage renal disease (Goldblum and Reed, Ann.Intern. Med. 93:597 (1980); Lahnborg et al, Transplantation 28:111(1979); Drivas et al, Invest. Urol. 17:241 (1979); Keane and Raij, In:Drukkar et al eds. Replacement of Renal Function by Dialysis, 2nd ed.,pp. 646-58 (1983)), acquired immunodeficiency syndrome (AIDS) (Bender etal, J. Infect. Disease 152:409 (1985), Smith et al, J. Clin. Invest.74:2121 (1984)), liver disease (Rimola, In: McIntyre et al eds OxfordTextbook of Clinical Hepatology, pp. 1272-84 (1991)) and diseases of thelung, including cystic fibrosis (Gomez and Schreiber, unpublishedobservations) and acute respiratory distress syndrome (ARDS) (Rossman etal, Clin. Res. 41:251A (1993)). Defective Fcγ receptor-dependentclearance has been observed in certain of these diseases. Thus, there isa clear need for methods that can be used to correct defective Fcγreceptor function and/or enhance functional Fc receptor expression andthereby stimulate host defense. The present invention provides suchmethods and compounds and compositions suitable for use therein.

OBJECTS AND SUMMARY OF THE INVENTION

It is a general object of the present invention to provide a method ofcombating infection by stimulating phagocytosis.

It is the specific object of the invention to provide a method ofstimulating phagocytosis by modulating the number and type of Fcreceptors present on cells that normally possess such receptors,including monocytes and macrophages. In addition, it is a specificobject of the invention to provide a method of combating infection byrendering cells phagocytic that do not normally possess that function,such as fibroblasts or epithelial or endothelial cells not normallyexpressing Fcγ receptors.

It is a further object of the invention to provide constructs suitablefor use in gene therapy protocols that encode Fc receptors, and cellstransformed therewith.

In one embodiment, the present invention relates to a method ofincreasing the phagocytic potential of cells present in a mammal thatcomprises introducing into the cells a DNA molecule coding for an Fcreceptor. The introduction is effected under conditions such that theDNA molecule is expressed, the Fc receptor produced, and the phagocyticpotential of the cells thereby increased.

In a further embodiment, the present invention relates to a method ofincreasing the phagocytic potential of cells of a mammal that comprises:

i) removing cells from the mammal,

ii) introducing into the cells a DNA molecule encoding an Fc receptor,and

iii) reintroducing the cells into the mammal under conditions such thatthe DNA molecule is expressed, the Fc receptor produced, and thephagocytic potential of the cells thereby increased. One skilled in theart will appreciate that steps (i)-(iii) can be carried out usingmethodologies known in the art.

In other embodiments, the present invention relates to a liposomecomprising a DNA molecule encoding an Fc receptor, a bacteriumcomprising a DNA molecule encoding an Fc receptor, a T cell comprisingan exogenous DNA sequence encoding an Fc receptor, and a B cellcomprising an exogenous DNA sequence encoding an Fc receptor.

In yet another embodiment, the present invention relates to a DNAconstruct encoding an Fc receptor comprising domains, or functionalportions thereof, from at least two of FcγRI, FcγRII and FcγRIII,wherein the domains, or portions thereof, are such that the receptorrenders cells phagocytic that comprise same. The invention also relatesto the encoded Fc receptor.

In a further embodiment, the present invention relates to a method oftreating an infection comprising administering to a mammal in need ofsuch treatment a DNA molecule encoding an Fc receptor. Theadministration is effected under conditions such that the DNA moleculeis expressed in cells of the mammal, the Fc receptor produced, and thephagocytic potential of the cells thereby increased. The resulting cellsphagocytose IgG-coated particles causing the infection, orIgG-containing soluble immune complexes derived from the infection.

Further objects and advantages of the present invention will be clearfrom the description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1--a) Biotinylation of D58 (Src+) and SAR6 (Src-) cells infectedwith FcγRIIA. Immunoprecipitation with anti-FcγRII mAb IV.3 demonstratesthe 40 kD FcγRIIA protein in the membrane of FcγRIIA-infected cells(lanes 2 and 4). No receptor is present in the sham-infected cells(lanes 1 and 3). b) Phosphorylation of FcγRIIA on tyrosine afterreceptor crosslinking in FcγRIIA-infected D58 and SAR6 cells.Phosphotyrosine containing proteins were immunoprecipitated from celllysates with and without FcγRIIA stimulating (+EA and -EA). Induction ofthe tyrosine phosphorylated 40 KD receptor is seen in lanes 6 and 8.

FIG. 2--Fluorescence histograms of (a) D58 and (b) SAR6 cells infectedwith FcγRIIA. The dotted line represents cells stained with an isotypecontrol mAB and the solid lines represent cells stained withanti-FcγRII.

FIG. 3--In vitro immune complex kinase assay of Src related tyrosinekinases from FcγRIIA infected D58 (Src+) (lanes 1-6) and SAR6 (Src-)cells (lanes 7-12). FcγRIIA-infected and sham-infected cells were lysedand cell lysates immunoprecipitated with the antibodies indicated aboveeach lane (RAM is the rabbit-anti-mouse control, IV.3 is anti-FcγRIImAb, Src and Fyn are mAbs specific for these kinases). Immune complexeswere exposed to [γ³² P]ATP to allow autophosphorylation of the kinasesand phosphorylation of FcγRIIA. The positions of the phosphorylated Src,Fyn and FcγRIIA proteins are indicated by the open squares, stars andarrows, respectively. Lanes 2 and 8, representing immunoprecipitateswith Src antibody alone, confirm the Src+ and Src- phenotypes of the D58and SAR6 cell lines.

FIG. 4--Macrophage Fcγ-receptor-mediated clearance of IgG-sensitizedradiolabeled red cells in patients with alcoholic cirrhosis of the liver(n=49), non-cirrhotic alcoholic subjects (n=10) and healthy volunteers.The middle three curves (means±SEM) represent values for clearance ofIgG-sensitized red cells in these 79 subjects; the upper pair of curves,the clearance of unsensitized autologous red cells in five patients andfive controls; and the lower pair of curves, the clearance ofheat-damaged red cells (heated for 30 minutes at 56° C.) in fivepatients and five controls.

FIG. 5--Macrophage Fcγ-receptor-mediated clearance of IgG-sensitizedradiolabeled red cells in patients with alcoholic cirrhosis of the liver(n=49), and healthy volunteers (n=20). The four middle curves(means±SEM) represent values for clearance in these 69 subjects:patients with mildly decompensated alcoholic cirrhosis of the liver(cirrhosis I, n=17), patients with moderately decompensated alcoholiccirrhosis of the liver (cirrhosis II, n=17), patients with severelydecompensated alcoholic cirrhosis of the liver (patients III, n=15), andcontrols (n=20).

FIG. 6--Macrophage Fcγ-receptor-mediated clearance of IgG-coated redcells (as half-time) in patients with alcoholic cirrhosis of the liver(n=49) and in controls (n=20). The half-time was significantly longer inthe eleven patients in whom severe infection developed during follow-up.

FIG. 7--Recognition of human IgG(anti-RhD)-coated red cells by monocytesfrom patients (n=49) and controls (n=20). IgG-sensitized, ⁵¹ Cr-labeled(2×10)⁷ erythrocytes were added to monolayers of monocytes, and thepercentage of red cells bound by monocytes was determined by measuringthe radioactivity. Values are means ±SEM.

FIG. 8--Recognition of mouse IgG2b-coated red cells by monocytes frompatients (n=49) and controls (n=20). IgG2b-sensitized erythrocytes wereadded to monolayers of monocytes, and the percentage of monocytesbinding >3 RBC per cell was determined. Values are means ±SEM.

FIG. 9--Macrophage Fcγ-receptor-mediated clearance in patients withcirculating immune complexes (n=7). The curves for these patients fellinto the range for the patient group.

FIG. 10--Tyrosine phosphorylation in wild type J32 and in mutant J32-3.2transfectants. Antiphosphotyrosine immunoblots were prepared followingimmunoprecipitation of cell lysates with either anti-phosphotyrosineantibody or anti-FcγRII antibody. The 40 kD FcγRII receptor isphosphorylated on tyrosine following FcγRII activation.

FIG. 11--Fluorescence histograms of J32/FcγRIIA and J32-3.2/FcγRIIAstable transfectants, and FcγRIIA expressing clones. Flow cytometry wasemployed with anti-FcγRII monoclonal antibody IV.3 or with an isotypecontrol (Indik et al, J. Clin. Invest. 88:1766 (1991)).

FIG. 12--Phagocytosis of IgG coated erythrocytes by J32 and J32-3.2transfectants. EA was prepared as described previously (Indik et al, J.Clin. Invest. 88:1766 (1991)), overlaid onto transfected orsham-transfected T-cells and incubated at 37° C. for 30 minutes. UnboundEA was removed by washing with PBS and extracellular bound EA wasremoved by exposure to hypotonic buffer before staining withWright-Geimsa.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods of modulating the phagocyticpotential of cells that are naturally phagocytic, such as macrophages,and to methods of rendering cells phagocytic that do not naturallypossess that function. In so doing, the present invention providesinnovative treatment regimens that can be used to combat infectionsassociated with various disease states.

Drug Induced Enhancement of Fcγ Receptor Expression

In one embodiment, the present invention relates to a method ofenhancing Fcγ receptor expression on phagocytic cells of a mammal,including macrophages. The method comprises administering to the mammalan active agent, such as the cytokine interferon gamma (IFN-γ), anestrogen or estrogen analog, or a hematopoietic growth factor such asgranulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophagecolony stimulating factor (M-CSF). IFN-γ has been shown to modulate thelevels of FcγRI and FcγRII apparently by increasing gene transcription.Dexamethasone has been reported to influence this IFN-γ-inducedenhancement of transcription in a cell-specific manner (Comber et al,Cell. Immunol. 145:324 (1992)). Estradiol and diethylstilbesterol havebeen shown to facilitate clearance of IgG-coated cells (Friedman et al,J. Clin. Invest. 75:162 (1985); Ruiz et al, Clin. Res. 38:367A (1990)).GM-CSF has been shown to selectively increase monocyte FcγRII expressionand function (Rossman et al, Exp. Hematol. 21:177 (1993)), and,similarly, M-CSF has been shown to increase splenic macrophage Fcγreceptors and thereby enhance the clearance of IgG-coated cells (Ruiz etal, Clin. Res. 40:796A (1992)).

One or more of the above-referenced active agents can be combined withan appropriate carrier to form a dosage form suitable for use in themethod of the present invention. The amount administered will varydepending on the patient, the agent, the clinical response sought andthe route of administration. Appropriate concentrations and dosageregimens can be readily determined by one skilled in the art havingknowledge of these agents.

The active agents can be formulated as capsules, tablets, and the like,and as solutions and suspensions suitable for intravenous or parenteraladministration. The agents can also be formulated as aerosols foradministration to the lung. Carriers used are pharmaceuticallyacceptable and depend on the dosage form.

In vivo synthesis of the above active agents can be effected, forexample, at a particular site, by introducing into cells of the patientsequences encoding the agent in an appropriate vector (e.g. anadenoviral or retroviral vector) preferably in combination with an Fcγreceptor encoding sequence (see below). In a preferred embodiment, thesequence encoding the agent encodes M-CSF and the sequence encoding thereceptor encodes the γ chain of FcγRIII. Such encoding sequences canalso be administered, for example, in liposomes, particularly where lungis the target tissue.

Conditions amenable to treatment by the above-noted active agentsinclude those characterized by reduced macrophage Fcγ receptor number orfunction, for example, chronic renal failure, liver disease andpulmonary disorders, including acute respiratory distress syndrome(ARDS), AIDS and cystic fibrosis. Such agents can be used in combinationwith one or more of the therapeutic approaches described below toenhance Fcγ receptor activity and thereby treat infections that oftenaccompany these conditions and others.

Fcγ Receptor Gene Therapy

In a further embodiment, the present invention relates to the use ofrecombinant and gene therapy protocols to modulate Fc receptorexpression. As noted above, genes encoding all three classes of Fcγreceptors have been isolated and cloned. All three receptor classes,FcγRI, FcγRII and FcγRIII, consist of distinct domains corresponding totheir location within the cell. The cDNA structure of the FcγRII classof receptors, for example, consists of a 5' untranslated region,sequences coding for a signal peptide region (S), an extracellulardomain (EC), a transmembrane region (TM), an intracytoplasmic domain(C), and a 3' untranslated region (Schreiber et al, Clin. Immunol.Immunopath., 62:S66 (1992), Cassel et al, Molec. Immunol. 30:451(1993)). Likewise, the predicted polypeptide sequence of FcγRI shows ahydrophobic signal sequence, a hydrophobic transmembrane region and acharged cytoplasmic domain, in addition to an extracellular region thatconsists of three immunoglobulin-like domains, two of which sharehomology with the other Fcγ receptors (Allen and Seed, Science 243:378(1989); Schreiber et al, Clin. Immunol. Immunopath., 62:S66 (1992)).FcγRIIIA is a complex consisting of a single α chain and a homo- orhetero- dimer of associated γ and ζ chains (Letourneur et al, J.Immunol. 147:2652 (1991); Ra et al Nature (Lond.) 241:752 (1989); Parket al, Clin. Res. 41:324A (1993)). Both the γ and ζ chains mediatephagocytosis, the γ chain being more efficient (Park et al, Clin. Res.41:324A (1993)). The extracellular domain of FcγRIII is closelyhomologous to that of FcγRI and FcγRII, however, the transmembranedomain of FcγIII terminates in a 200-220 residue hydrophobic domainfollowed by four hydrophobic residues, one of which is charged (Simmonsand Seed, Nature 333:568-570 (1988)). FcγRIII thus differs from FcγRIand FcγRII in that the latter two have substantial intracellularcytoplasmic domains.

FcγRI is unique among the three classes of human Fcγ receptors not onlyin its high affinity for IgG but also in the structure of itscytoplasmic domain. Macrophage FcγRII and the γ chain of FcγRIII havetyrosine residues in their cytoplasmic domains that are required forphagocytosis. In contrast, FcγRI does not contain tyrosine residues inits cytoplasmic domain (Allen and Seed, Science 243:378 (1989)) and isnot phosphorylated on tyrosine. Further, FcγRI is unusual among the Iggene family of receptors in not requiring its cytoplasmic domain forphagocytosis (Indik et al, Clin. Res. 41:170A (1993)).

Recombinant techniques make it possible to manipulate the domains ofnaturally occurring receptors and thereby design Fc receptors havingspecific characteristics. The present invention contemplates the use ingene therapy regimens of DNA sequences encoding such selectivelyconstructed receptors, comprising domains from single or multiple Fcγreceptors, to effect the production of receptors having definedactivities, both in cells that normally produce Fcγ receptors and incells that normally do not. In the former case, the Fc receptor sequenceintroduced into target cells can encode a protein essentially identicalto that normally produced by the cell. Alternatively, the sequenceintroduced can encode: i) an Fc receptor protein that is functionallycomparable to, but structurally different from, the naturally occurringreceptor (e.g. a protein comprising only functional portions of thedomain(s) (for example, the cytoplasmic domain) of the naturallyoccurring receptor), or ii) a receptor protein that differs functionallyand structurally from the Fc receptor that is normally present on thecell (e.g. a chimeric receptor protein comprising a high affinity FcγRIextracellular domain and transmembrane and cytoplasmic domains fromFcγRIIA or FcγRIIIA). The present invention thus makes it possible tocompensate for deficiencies in the production of Fc receptors of aparticular functional type, which deficiencies may occur in associationwith a particular disease state. The invention also makes it possible tomanipulate the composition of the Fc receptor population of a particularcell type. That is, a cell producing predominantly high affinityreceptors can be engineered so as to produce predominantly low affinityFc receptors.

Equally important, the present invention makes it possible to rendercells phagocytic that do not normally possess that function. Sequencesencoding naturally occurring Fcγ receptors or sequences encodingnon-naturally occurring Fc receptors, for example, chimeric receptorsthat include entire domains, or functional portions thereof, from two ormore naturally occurring Fcγ receptors, can be introduced into suchcells. The chimeric receptors can be designed so as to take into accountboth the phagocytic potential of the cells into which the encodingsequences are to be introduced and the receptor domain properties suitedfor achieving the desired therapeutic effect. While not all cells areequally suitable as recipients for all Fc receptor-encoding constructs,operability can be readily assessed using in vitro model systems such asthose described by Indik et al (J. Clin. Invest. 88:1766 (1991) andHunter et al, Clin. Res. 41:244A (1993); see also Amigorena et al,Nature (Lond) 358:337 (1992); Park et al, Clin. Res. 41:324A (1993);Toijman et al, Blood 79:1651 (1992); Kruskal et al, J. Exp. Med.176:1673 (1992); (see also Examples below)). This embodiment of theinvention may be particularly advantageous since cells, such asfibroblasts, that are rendered phagocytic may injest particles withoutreleasing significant quantities of superoxide radicals or toxicbiologically active products. This is in contrast to cells that arenormally phagocytic, such as macrophages. One skilled in the art willappreciate that a reduction in the release of toxic products results ina reduction in the possibility of inflammation.

Constructs:

Chimeric Fc receptors suitable for use in the present invention includethose prepared as detailed in the Examples below. For instance, singlechain chimeras of the α and γ chains of FcRIIIA can be prepared.Sequences encoding such chimeras have been introduced into COS-1 cellsand the phagocytic potential conferred examined. For example, a DNAsequence encoding the extracellular domain of the α chain of FcγRIIIA,the transmembrane domain of the γ chain of FcγRIIIA or FcγRI and thecytoplasmic domain of the γ chain of FcγRIIIA has been transfected intoCOS-1 cells (the transmembrane domain of the α chain of FcγRIII can beused in lieu of that of the γ chain, though perhaps not as effectively).Such chimeras display phagocytic activity in the COS-1 assay systemthough not at a level equivalent to the multichain form of FcγRIIIA. Inspite of the reduced activity, single chain constructs are clearlyadvantageous in view of the difficulties inherent both in introducinginto target cells multiple sequences and in achieving propercomplexation of the encoded proteins.

Fc chimeric receptors have also been prepared from a combination ofdomains of FcγRII isoforms and from a combination of FcγRI and FcγRIIdomains. Specifically, a chimeric receptor comprising the extracellularand transmembrane domains of FcγRIIB2 and the cytoplasmic domain ofFcγRIIA has been shown to confer phagocytic potential on host cells,thus demonstrating that the FcγRIIB2 transmembrane domain is capable oftransmitting the phagocytic signal to the FcγRIIA cytoplasmic domain(FcγRIIB receptors do not themselves confer phagocytic potential).Similarly, a chimeric receptor comprising the extracellular domain ofFcγRI and the transmembrane and cytoplasmic domains of FcγRIIA has beenshown to induce phagocytosis in host cells. In contrast, chimerascomprising the extracellular domain of FcγRI and the transmembranedomain of FcγRI or FcγRIIA do not result in phagocytosis when thecytoplasmic domain is from FcγRIIA or FcγRI, respectively. However,chimeras comprising the extracellular domain of FcγRI, the transmembranedomain of FcγRI and the cytoplasmic domain of the γ chain of FcγRIII, doresult in phagocytosis. It will be appreciated that chimeras comprisingthe extracellular domain of FcγRI (and appropriate transmembrane andcytoplasmic domains) can be advantageous in view of the high bindingaffinity of the FcγRI extracellular region.

Chimeras in addition to those described above and detailed below arecontemplated. For example, the cytoplasmic domain of FcγRIIA can be usedin combination with the extracellular domain of FcγRI and thetransmembrane domain of FcγRIIA. Further, the extracellular andtransmembrane domains of FcγRI or FcγRII can be used in combination withthe cytoplasmic domain of the γ chain of FcγRIII. Further, chimeras ofthe invention can include the extracellular domain from FcγRIIA, FcγRIor from the α chain of FcγRIII, the transmembrane domain from FcγRIIA orfrom the α or γ chain of FcγRIII, and the cytoplasmic domain of eitherthe γ chain of FcγRIII or FcγRIIA (e.g., i) the extracellular andtransmembrane domains of FcγRIIA, ii) the extracellular domain of the αchain of FcγRIII and the transmembrane domain of the γ chain of FcγRIII,or iii) the extracellular domain of FcγRI and the transmembrane domainof the α or γ chain of FcγRIII--each with the cytoplasmic domain fromeither the γ chain of FcγRIII or FcγRIIA (it is noted that preliminaryresults suggest that certain chimeras comprising the transmembranedomain of the α chain of FcγRIII may not be operative).

While chimeras of the invention can include the entire extracellular,transmembrane and cytoplasmic domains of the respective naturallyoccurring receptors, such is not necessarily the case. Rather, thechimeras can comprise only the functional portion(s) of the respectivedomains. For example, in the case of the cytoplasmic domain of FcγRIIA,truncation at amino acid 303 (which results in deletion of the terminal8 amino acids but preservation of the two tyrosine (Y282 andY298)-containing core sequences important in phagocytosis does notdecrease phagocytosis (Mitchell et al, Clin. Res. 41:1894A (1993)).Truncation of the FcγRIIA cytoplasmic domain at amino acid 268 or 280,however, results in receptors lacking the tyrosines at positions 282 and288, and lacking phagocytic activity. These data are consistent with theimportance of tyrosine residues in the cytoplasmic Fc receptor domain intransmission of the cytoplasmic signal. In treatment regimens in whichsuppression of phagocytic potential is advantageous (for example,autoimmune diseases) these later mutants or peptides derived from ormimicking these mutants can be useful (see the commonly ownedapplication entitled "Method of Inhibiting Phagocytosis" filedconcurrently herewith, the entire disclosure of which is incorporatedherein by reference). It will be appreciated, however, that whenpotentiation of phagocytosis is sought, functionality of each of thedomains must be preserved. In this regard, it appears that the secondYX2L of the core sequence of the cytoplasmic domain of FcγRIIA(E-X8-D-X2-Y-X2-L-X12-Y-X2-L) (SEQ ID NO:5) and the γ chain of FcγRIIIA(D/E-X2,7-D/E-Y-X2-L-X7-Y-X2-L) (SEQ ID NO:6) are particularly importantfor phagocytosis (note also that the exon 3 domain of the γ chain ofFcγRIII that is 5' or amino terminal to the Y-X2-L (SEQ ID NO:7) motifappears to play a role in phagocytosis since its elimination diminishesphagocytosis by the γ subunit of FcγRIIIA) (the numbers following theletter X denote the number of amino acids at that position; X can be anyamino acid but X within a Y-X2-L preferably represents the amino acidspresent in the Y-X2-L sequence of the cytoplasmic domain of FcγRIIA orthe γ chain of FcγRIII). Accordingly, it can be expected thatphagocytosis can be increased by multiplying the number of copies of thecore sequence, for example, in FcγRIIA or in the γ chain of FcγRIIIA, orby multiplying the number of copies of the second Y-X2-L present inthose core sequences. The specific amino acids in this second Y-X2-L areimportant for phagocytosis and appear to provide specificity to thephagocytic signal. It is also expected that phagocytic activity can beincreased (as compared to the wild type gamma chain) by, for example,inserting the FcγRIIA second Y-X2-L into the γ chain of FcγRIIIA (ascompared to the wild type gamma chain). Furthermore, it is expected thatinserting the second cytoplasmic domain Y-X2-L of the γ chain ofFcγRIIIA (or both the first and second cytoplasmic domain Y-X2-L of theγ chain) into the ζ chain of FcγRIIIA will increase the phagocyticactivity of the ζ chain. Further, the inclusion of two additional Y-X2-Lor Y-X3-I motifs to FcγRIIB (which itself is non-phagocytic) rendersthis receptor phagocytic (this includes adding a variation of theY-X2-L, Y-X3-I, to the carboxyterminal portion of the cytoplasmicdomain). As indicated above, fibroblasts and fibroblast-like cells (forexample, COS cells) can be used to assess the operability of aparticular receptor construct.

The above-described chimeras of the invention can be constructed by thepolymerase chain reaction (PCR) (Horton et al, Biotechniques 8:528(1990)) using as templates appropriate receptor cDNA and appropriateoligonucleotides. PCR products can be directly cloned into an expressionvector, for example, pSVL, and confirmed by complete sequencing. Theexpression of the chimeric receptors can be assayed by flow cytometryusing anti-Fcγ receptor mAbs and phagocytic function can be evaluatedfollowing incubation of IgG-sensitized RBCs.

More specifically, two step overlap extension PCR, a technique thatallows introduction of mutations into any part of a PCR fragment, can beused to generate the chimeric molecules of the invention, as well as themutated/truncated receptors described herein. In the first step inoverlap extension PCR, two primer pairs, 1a and 1b and 2a and 2b, areused to generate two overlapping fragments, 1 and 2. In step 2, whenthese two fragments are mixed, denatured and reannealed, the 3' end ofthe sense strand of fragment 1 anneals to the 3' end of the antisensestrand of fragment 2. This overlap can be extended to form the entirerecombinant product and can be amplified by PCR using primers 1a and 2b.The overlap region is determined by primers 1b and 2a and can containany sequence as long as parts of the oligomers are complementary. Thisregion is where base changes are incorporated when the technique is usedfor site directed mutagenesis. Alternatively, the overlap can bedesigned to make a clean joint between two fragments from two differentDNA molecules to form a chimeric molecule. For construction of chimericmutants, primers 1b and 2a are designed to contain regions from bothcontributing molecules so that fragments 1 and 2 can anneal. Forexample, to construct the chimera containing the FcγRIIIAα extracellularregion and the transmembrane and cytoplasmic domains of the γ chain, thefollowing 2 pairs of oligomer primers are used (primer 1b is shown3'-5'):

1a.5'ACGATGTCTAGAGGTGACTTGTCCACTCC3'(SEQ ID NO:1)(sense)

1b.3'GGTGGACCCATGGTTGAGACGATATAGGAC5'(SEQ ID NO:2)(antisense)

2a.5'CCACCTGGGTACCAACTCTGCTATATCCTG3'(SEQ ID NO:3)(sense)

2b.5'ATGGCGAGCTCTCCGGTAAACAGCATCTGAG3'(SEQ ID NO:4)(antisense)

Xbal and Sacl restriction sites can be introduced in primers 1a and 2brespectively so that the final PCR product encoding the chimericreceptor can be ligated in the proper orientation into, for example, anSV40 based expression vector (e.g., PSVL) restricted with Xbal and Sacl.To produce truncated molecules, stop codons can be introduced viaprimers 1b and 2a. In a similar fashion, tyrosine codons can be replacedby phenylalanine codons and serine or threonine codons by alaninecodons.

Target cells and modes of administration:

As noted above, the present invention can be used to treat patients thatare predisposed to an increased risk of infection. Such patientsinclude, but are not limited to, those suffering from liver diseaseresulting, for example, from alcoholic cirrhosis, from kidney disorders,such as end-stage renal disease, and from pulmonary disorders includingcystic fibrosis and ARDS. AIDS patients are also appropriate candidatesfor treatment in accordance with the present invention. In eachinstance, treatment is effected by increasing the phagocytic potentialof cells of the patient.

In the case of pulmonary disorders, the receptor-encoding sequence canbe administered to the cells of the lung, including macrophages, in theform of an aerosol. The encoding sequence can be present in the aerosolas a particle (e.g. liposome or non-infectious bacteria, for example,Listeria) that is phagocytosed by the pulmonary macrophages. Theencoding sequence can also be present in a viral vector.

Viral vectors can also be used to introduce the Fc receptor-encodingsequence of the invention into cells of the pulmonary tree, includingfibroblasts, epithelial cells and other cells present in the lung. Thevectors can be introduced as an aerosol and can take the form of areplication defective herpes or adenoviral vector. Retrovital vectorscan also be used, as well as other viral vectors. (See, generally,Bajocchi et al, Nat. Genet. 3:229 (1993); Lemarchand et al, Circ. Res.,72:1132 (1993); Ram et al, Cancer Res. 53:83 (1993); Crystal, Am. J.Med. 92:44s (1992); Yoshimura et al, Nucl. Acids Res. 20:3233 (1992);Morecki et al, Cancer Immunol. Immunother. 32:342 (1991); Culver et al,Hum. Gene Ther. 1:399 (1990); Culver et al, Transplant. Proc., 23:170(1991)).

The Fc receptor-encoding sequences of the invention can also beintroduced into cells such as T cells thereby rendering them phagocytic.The advantages of phagocytic T cells are clear, particularly incombating infections that accompany diseases such as AIDS. The abundanceof T cells is such that by transforming them with the Fc receptorencoding sequences of the invention, the phagocytic capacity of theblood is substantially increased.

T cells can be rendered phagocytic by transforming them in vitro with,for example, a viral vector containing a sequence encoding an Fcreceptor (e.g. FcγRIIA). Techniques such as electroporation can also beused. The transformed T cells can then be reintroduced into the patientfrom which they were derived. Example X details the transformation ofT-cells with FcγRIIA and the results presented demonstrate thatphagocytic activity is conferred on these cells. In addition, FcγRIIA isphosphorylated in the T-cells when activated, similar to thephosphorylation observed in activated monocytes and macrophages. FcγRIIAactivation in these T-cells leads to tyrosine kinase activation andphosphorylation. The T-cell tyrosine kinase ZAP-70 is activated(phosphorylated) upon FcγRIIA activation in T-cells. B lymphocytes areless abundant than T lymphocytes, but they too can be renderedphagocytic using similar protocols (see Example VII).

Further, blood monocytes can be transformed ex vivo with thereceptor-encoding sequence of the invention (using, for example,physical techniques such as electroporation, or vectors, including viralvectors (e.g., retrovital vectors, adenoviral vectors, or herpes viralvectors); liposomes and Listeria can also be expected to be useful intransforming monocytes and then reintroduced into the patient). Thisprotocol is particularly advantageous when the liver or spleen is thetarget site.

In addition to the above, the present invention can be used withpatients suffering from immune complex diseases such as lupuserythematosus and rheumatoid arthritis to increase local clearance ofcirculating immune complexes so as to prevent their deposition intissues, such as the kidney, and in the joints. This increase can beeffected by stimulating liver and splenic macrophage phagocyticpotential using protocols such as those described herein.

It will be appreciated from a reading of the foregoing that, dependingon the target cell and the effect sought, various methods can be used tointroduce receptor-encoding sequence into the cell (in addition toelectroporation noted above, calcium phosphate as well as othertechniques can be used to introduce naked DNA). It will also beappreciated that the gene therapy approach to enhancing phagocyticpotential can be used alone or in combination with the drug therapyapproach described above. The combination therapy makes it possible toincrease the number of naturally occurring receptors and at the sametime effect the selective expression of receptors of a particularfunctional type.

The following non-limiting Examples describe certain aspects of theinvention in greater detail.

EXAMPLE I In Vivo Administration of hrM-CSF Increases Splenic MacrophageFcγ Receptors

Human recombinant macrophage colony stimulating factor (hrM-CSF) wasstudied in vivo using an established model in the guinea pig (Schreiberet al, J. Clin. Invest. 51:575 (1972)). Adult male guinea pigs weretreated for 5 days with hrM-CSF (500 μg/kg) and splenic macrophage FcγRfunction and protein expression were assessed by i) the splenicmacrophage clearance of IgG sensitized ⁵¹ Cr-guinea pig RBC (EA), ii)the in vitro binding of EA by isolated splenic macrophage, and iii) FACSanalysis using monoclonal antibodies with specificity for the two guineapig splenic macrophage Fcγ receptors, FcγR1,2 and FcγR2. Treatment withhrM-CSF enchanced the clearance of EA by 72 ±5%. In addition, a greaterproportion of isolated splenic macrophages from hrM-CSF treated animalsbound EA in vitro: 80±7% vs 48±4%(sham), p<0.001. In vivo hrM-CSFincreased the expression of both splenic macrophages Fcγ receptors:81±6% and 130±10% for FcγR1,2 and FcγR2, respectively. The lowesteffective dose of hrM-CSF was 250 μg/kg, increasing the expression ofFcγR1,2 by 26±3% and FcγR2 by 42±4%. At this dose, the clearance of EAwas also enhanced. The effect of hrM-CSF required at least 4 days oftreatment.

EXAMPLE II FcγIIA Mediates Phagocytosis and Receptor Phosphorylation ina Fibroblast Cell Line

Experimental Protocols:

Cell culture and reagents:

The SAR6 cell line was derived from primary embryonic mouse fibroblastsin which both Src alleles had been disrupted by homologous recombinationusing the neomycin resistance gene (Thomas et al, Science 254:568(1991)). D58 was derived from primary embryonic mouse fibroblasts thatwere wild type for Src. Cells were maintained in DMEM containing glucose(4.5 mg/ml), glutamine (25 mg/ml), penicillin (100 U/ml), streptomycin(100 μg/ml) and 10% heat inactivated fetal calf serum.

Retroviral infections:

FcγRIIA was inserted into the HindIII site of the retroviral vector pLCX(Miller and Rosman, Biotechniques 9:908 (1989)) under control fo the CMVpromoter. The resulting construct, pLNCX2A, was transfected into theecotropic packaging cell line, Psi2. Two days after transfection, thecells were diluted 1:20 and G418 resistant colonies were isolated andassessed for virus production. The stock gave 1×10 G418 resistantcolonies per milliliter. 0.1 ml of viral stock was used to infect DS8and SAR6 cells (2.5×10 cells per infection). Twenty four hours afterinfection, the cells were diluted 1:3 and allowed to reach 80-90%confluence before assaying for cell surface expression of FcγRIIA andfor phagocytosis. Transient infections were carried out due to the factthat the G418 resistant phenotype of the SAR6 cell line prohibited theselection of stable lines using this retroviral vector.

Flow cytometry:

To determine the extent of FcγRIIA expression on the cell surface ofinfected D58 and SAR6 cells, samples were stained withfluorescein-labeled anti-FcγRII mAb (IV.3) or with an isotype control(Indik et al, J. Clin. Invest. 88:1766 (1991)). Fluorescence wasmeasured on a FACStar (Becton-Dickinson, Mountainview, Calif.). 10,000events were analysed in each case and mean fluorescence intensities wereestimated and contour maps were generated using Consort 30 software.

Binding and phagocytosis of IgG-sensitized sheep red blood cells (EA): CEA was prepared as described previously (Indik et al, J. Clin. Invest.88:1766 (1991)), overlaid onto the infected cells and incubated at 37°C. for 30 minutes. Unbound EA was removed by washing with PBS and theplates stained with Wright-Geimsa to assess rosetting. To determinephagocytosis, extracellular bound EA was removed by exposure tohypotonic buffer before staining with Wright-Geimsa.

Biotinylation of cell membranes:

Twenty four hours after infection, FcγRIIA-infected and sham-infectedSAR6 and D58 cells were plated on 100 mm petri dishes. After a furthertwenty four hours, the cells (2×10) were washed once with PBS, overlaidwith 1.0 ml of PBS containing 100 μl of 1M NaHCO and 100 ml of 1 mg/mlbiotin (Pierce, Rockford, Ill.) and incubated at room temperature for 60minutes. One hundred μl of NHCl was added and incubation continued for afurther 10 minutes. The cells were washed once with PBS and lysed with1.0 ml RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS,158 mM NaCl, 10-mM Tris pH7.2, 5 mM NaEGTA, 1 mM phenylmethylsulphonylfluoride, 1 mM NaVO) at 4° C. for 30 minutes. FcγRIIA wasimmunoprecipitated from the biotinylated cell membrane extract with antiFcγRII mAb (Eisman and Bolen, Nature 355:78 (1992)) and analyzed on a7.5% SDS-polyacrylamide gel (PAGE). Immunoblots were probed withavidin-horseradish peroxidase (BioRad, 1:1000 dilution), followed byEnhanced Chemiluminescence reagents (Amersham Corp.) and visualizedusing Kodak XAR-5 film.

Phosphotyrosine immunoblots:

FcγRIIA-infected and sham-infected D58 and SAR6 cells (2×10 cells per100 mm petri dish) were overlaid with 500 μl EA and incubated at 37° C.for 30 minutes to activate FcγRIIA. After washing with PBS to removeunbound EA, the bound EA was removed by exposure to hypotonic buffer.Cells were lysed on the plates with 1.0 ml RIPA buffer at 40° C. for 30minutes and phosphotyrosine containing proteins were immunoprecipitatedfrom the cell lysates using polyclonal rabbit antisera UP28 (Huang etal, J. Biol. Chem. 267:5467 (1992)). The immunoprecipitates wereanalyzed on a 7.5% SDS-PAGE and immunoblots probed withantiphosphotyrosine mAb, 4G10 (Huang et al, J. Biol. Chem. 267:5467(1992)).

In vitro immune complex kinase assay of Src-family protein tyrosinekinases from FcγRIIA infected Src- and Src+ cells:

FcγRIIA-infected and sham-infected SAR6 and D58 cells (2×10 cells per100 mm petri dish), were lysed with 1.0 ml RIPA buffer at 4° C., for 30minutes. Immunoprecipitations were performed by mixing cell lysates withthe following mAbs singly or in combination: anti-Src (Lipsiche et al,J. Virol. 48:352 (1983)), anti-FcγRII (Rosenfeld et al, J. Clin. Invest.76:2317 (1985)), anti-Fyn (Huang et al, J. Biol. Chem. 267:5467 (1992))and rabbit anti-mouse (RAM) IgG. The immune complexes were incubatedwith [γ³² P]ATP to allow autophosphorylation of the kinases andphosphorylation of the substrate and were separated by SDS-PAGE. The gelwas washed with 1N KOH at 55° C. for two hours to removeserine/threonine phosphorylation (tyrosine phosphorylation is relativelyresistant to alkali) before exposure to Kodak XAR-5 film.

Results of Phagocytosis and Phosphorylation Studies

Forty eight hours after infection of cell lines D58 and SAR with aretroviral vector containing a FcγIIA encoding sequence, cell surfacebiotinylation followed by immunoprecipitation with anti-FcγRII mAbdemonstrated that the 40 kD receptor was present in the membrane of bothSrc+ and Src- cells (FIG. 1a). Fluorescence histograms of FcγRIIAinfected SAR6 and D58 cells are shown in FIG. 2. In this representativeexperiment, sixty five percent of cells expressed the receptor in SAR6and eighty one percent in D58 with mean fluorescence intensities ofninety five and one hundred and fifty one, respectively. Both Src- andSrc+ cells incubated with IgG sensitized cells (EA) bound andphagocytosed these immune complexes. Forty three percent of cellsphagocytosed EA in the Src- mutant and seventy percent in D58. Incontrast, no binding or phagocytosis was observed in sham infectedcells.

To determine if the activated receptor was phosphorylated in the Src-cell line, phosphotyrosine containing proteins were immunoprecipitatedfrom activated and unactivated SAR6 and D58 infected cells. Crosslinkingof FcγRIIA with EA resulted in tyrosine phosphorylation of the 40 kDFcγRIIA receptor protein in both Src+ and Src- cells (FIG. 1b).

Although Src is not responsible for phosphorylating FcγRIIA in SAR6cells, FcγRIIA in these mouse fibroblasts was able to act as a substratefor Src related tyrosine kinases. An in vitro immune complex kinaseassay was performed on lysates from SAR6 and D58 cells that had beeninfected with FcγRIIA. Lysates were co-immunoprecipitated withantibodies specific for the receptor protein and with antibodiesspecific for either Src or Fyn kinases (FIG. 3). Theco-immunoprecipitates were incubated with [γ³² _(p]) ATP to allowautophosphorylation of the kinase and phosphorylation of FcγRIIA.FcγRIIA was phosphorylated by Src in this in vitro assay (FIG. 3, lane5). Fyn could also phosphorylate FcγRIIA, although to a lesser extentwhen compared to Src (lane 6). In the absence of the kinases, nophosphorylation of FcγRIIA was observed (lanes 4 and 10) consistent withthe lack of tyrosine kinase sequences in the receptor. In the Src-lysates, co-immunoprecipitation with Src and FcγRIIA did not result inphosphorylation of the receptor (lane 11), but a low level ofphosphorylation of FcγRIIA was observed in co-immunoprecipitates ofFcγRIIA and Fyn (lane 12). This may reflect the efficiency ofphosphorylation of the receptor by Fyn; alternatively the fibroblastsmay express different amounts of the two kinases.

EXAMPLE III High Affinity Fcγ Receptor (CD64) Induces Phagocytosis inthe Absence of its Cytoplasmic Domain

Wild type (WT) and a mutant (MT) FcγRI, engineered to omit thecytoplasmic domain (CYT), were transfected into COS cells and murinemacrophages (P388D1). The phagocytic potential of the transformed cellswas assessed using IgG-coated RBCs (EA) and RBCs conjugated with Fabanti-human FcγR1 mAb (E-mAb). FcγR1, in contrast to FcγRII, did notinduce phagocytosis in COS cells (assessed by electron microscopy) butdid induce a Ca²⁺ signal which required its CYT. However, both WT and MTFcγRI induced phagocytosis in P388D1. Phagocytosis by WT FcγRI wasinhibited by the tyrosine kinase inhibitor tyrphostin 23. Furthermore,activation of FcγRI on monocytes with Fab anti-FcγRI induced tyrosinephosphorylation of FcγRII, determined by anti-phosphotyrosineimmunoblots. FcγRI thus mediates a Ca²⁺ signal through its cytoplasmicdomain but not phagocytosis. FcγRI induced phagocytosis thereforerequires elements, present in macrophages but absent in COS cells, thatpermit transmembrane communication.

EXAMPLE IV Structural Requirements of the Human Fc Receptor FcγRIIA inPhagocytosis

The structural requirements of FcγRIIA in phagocytosis were examinedusing COS-1 cells, which lack endogenous Fc receptors, as the recipientin transfection studies. FcγRIIA has two (Y282 and Y298)tyrosine-containing core sequences, Y-X2-L, within a cytoplasmic motifsimilar to that in other Ig gene family receptors. Truncation of thecytoplasmic domain at amino acid 268 or 280, to produce mutants lackingboth these tyrosines and both core sequences, eliminated phagocyticactivity even though these transfectants bound IgG-sensitized cellsefficiently. Truncation at amino acid 303, deleting only the terminal 8amino acid and preserving both core sequences, did not decreasephagocytosis. Substitution of Y282 with phenylalanine (F) inhibitedphagocytosis and substitution of Y298 with F partially diminished thephagocytic signal. Substitution with F of the third cytoplasmic tyrosine(Y275) outside the conserved motif did not alter phagocytosis.Replacement of Y282 or Y298 with lysine reduced phagocytosis further,but replacing Y275 with lysine had little effect. Replacement by F ofeither Y275 or Y298 in combination with Y282 completely eliminatedphagocytic function, suggesting that they interact with Y282 intransmission of the signal. In contrast, some phagocytic activity waspreserved in mutants containing Y282, but with F at Y275 and Y298.Deletion of T284-L285 within the Y282MTL core sequence also diminishedphagocytosis. The two core Y282-X2-L and Y298-X2-L sequences contain anintervening stretch of amino acids with 2 prolines suggesting anintervening non-helical structure. A mutant, Δ287-291, in which 5 aminoacids including the 2 prolines were deleted reduced phagocytic function.The initial core cytoplasmic sequence Y282MTL and the proline containingregion between Y282 and Y298 are important for transmission of thephagocytic signal by FcγRIIA.

EXAMPLE V The Structure of the γ chain Fc Receptor Subunit DeterminesPhagocytic Function of Macrophage FcγRIII (FcγRIIIA)

A FcγRIIIA encoding sequence was transfected into COS-1 cells to studyits phagocytic function, determined by electron microscopy, in theabsence of other Fc receptors. Co-transfectants of FcγRIIIA-α witheither γ or ζ gave equivalent cell surface expression and binding ofIgG-coated cells (EA), but γ was 6 fold more effective than ζ inphagocytosis. To delineate the region of the γ chain important inphagocytosis, two deletion mutants, were constructed, deleting theC-terminal 7 amino acids or deleting the C-terminal 22 amino acids whichhave a tyrosine containing conserved motif, Y-X2-L-X7-Y-X2-L, present inseveral Ig gene super family receptors. The C-terminal 7 amino aciddeletion demonstrated minimally reduced phagocytic activity, whereas themore extensive deletion completely eliminated phagocytosis, suggestingthe importance of the conserved cytoplasmic motif. The role of theconserved cytoplasmic tyrosines was then examined. Conservativesubstitution by phenylalnine of either of the 2 cytoplasmic tyrosines inthe γ chain significantly decreased Ca²⁺ signaling and reducedphagocytosis by >99%. Tyrophostin 23 which alters tyrosine kinaseactivity reversibly inhibited phagocytosis, indicating thatphosphorylation of γ and/or downstream protein tyrosine kinase(s) isrequired for a phagocytic signal. Further, single chain Fcγ receptorchimeras, consisting of the γ cytoplasmic domain and the α extracellulardomain with the transmembrane domain of either FcγRIIIA-γ or FcγRI wereable to mediate a phagocytic signal. However, single chain chimeras werenot sufficient for full phagocytic activity.

EXAMPLE VI Examination of Phagocytosis by Chimeric Fcγ Receptors

FcγRIIA avidly binds and phagocytoses IgG-sensitized cells (EA), asassessed by electron microsopy using the COS cell transfection modelsystem, but FcγRI and two other FcγRII isoforms, FcγRIIB1 and FcγRIIB2,do not transmit a phagocytic signal although they also bind EA avidly.Chimeric receptors of FcγRI and FcγRII were constructed in order tofurther assess the function of their transmembrane and cytoplasmicdomains in phagocytosis. Chimeric transfectants consisting of theextracellular (EC) and transmembrane (TM) regions of FcγRIIB2 and thecytoplasmic domain (CYT) of FcγRIIA and chimeric transfectantsconsisting of the EC of FcγRI and the TM and CYT of FcγRIIA wereefficient in phagocytosis. In contrast, phagocytosis was greatlydiminished by chimeras consisting of the EC and TM of FcγRI and the CYTof FcγRIIA. In addition, a chimeric transfectant bearing the EC fromFcγRI, the TM from FcγRIIA and the CYT from FcγRI did not phagocytoseEA. These studies indicate that in this system: i) the transmembranedomain of FcγRIIB2 is able to provide the necessary structure to permita phagocytic signal by the cytoplasmic domain of FcγRIIA, ii) thetransmembrane domain of FcγRI is unable to transmit a phagocytic signalto the cytoplasmic domain of FcγRIIA, and iii) the transmembrane domainof FcγRIIA is unable to confer phagocytic competence to FcγRI.

EXAMPLE VII B-Cell Antigen Receptor Subunit Ig-γ Mediates PhagocyticSignal

The B-cell receptor complex is composed of an antigen recognitionsubunit noncovalently associated with a membrane subunit consisting ofheterodimers of two chains, Ig-α and Igβ/γ, which are products of themb-1 and B29 genes. Both membrane Ig subunits contain within theircytoplasmic regions a conserved sequence implicated in intracellularsignalling. Using COS cell transfectants, the Fc receptor FcγRIIA, whichis not present in B-cells, has been shown to mediate a phagocytic signaland to contain within its cytoplasmic domain a sequence similar in someaspects to that of Ig-α. Therefore, a FcγRIIA and Ig-α chimera wasconstructed, consisting of the extracellular and transmembrane domainsof FcγRIIA and the cytoplasmic domain of Ig-α. This chimeric receptorwas expressed in COS-1 cell transfectants, determined by flow cytometry,and bound IgG-sensitized RBCs (EA) efficiently. Furthermore,transfection of this chimeric receptor into COS-1 cells conferredphagocytic competence to COS-1 cells similar in extent to transfectionof the receptor FcγRIIA.

EXAMPLE VIII Alterations in Monocyte/Macrophage Fcγ Receptor Expressionin the Acute Respiratory Distress Syndrome (ARDS)

Monocytes from patients with ARDS were used to examine potentialalterations in Fcγ receptor expression. Since macrophages may expressall 3 classes of Fcγ receptors, specific mAbs for each class of Fcγreceptor and flow cytometry were used to quantitate Fcγ receptorexpression. Patients with ARDS met the following four criteria: i) acutebilateral alveolar-type infiltrates on chest radiograph, ii) severehypoxemic respiratory failure with PaO/FiO</=150 without PEEP, iii)absence of congestive heart failure, and iv) having a presumedpre-disposing cause of ARDS. Seven patients with ARDS were compared to 5normal controls. Whether measured as percent of cells expressing the Fcγreceptor or the difference in mean fluorescence intensity (MFI), FcγRIwas reduced in patients with ARDS (ARDS=36.0±6.3% [mean±SEM] or 22.6±7.0MFI; normal=52.8±11.3% or 35.6±6.4 MFI) and FcγRIII was increased(ARDS=15.6±7.9% or 12.1±4.9 MFI; normals=0.8±0.6% or 1.4±1.2(MFI). Nocorrelation was observed between decreased FcγRI and increased FcγRIIIexpression, suggesting differential regulation of these receptors invivo. No significant change was observed in the expression of FcγRII.Four of seven patients with ARDS died. One patient was restudiedfollowing recovery and Fcγ receptors returned to normal values.

EXAMPLE IX Fc Receptor Defect in Patients with Liver Disease

Experimental Protocols:

Patients:

Forty nine patients (16 women and 33 men) whose mean (±SD) age was55.2±8.3 years were studied. All patients had biopsy proven alcoholiccirrhosis of the liver and were followed up to a minimum period of twoyears after study: six died within this period. Ten alcoholicnon-cirrhotic subjects (4 women and 6 men; age 45±7 years) and, 20healthy volunteers (6 women and 14 men; age 52±12 years) served asconcurrent controls. Patients were classified in three groups accordingto their degree of liver insufficiency as assessed by the Orrego index(Orrego et al, Gastroenterology 76:105 (1979)).

Study Protocol:

Blood was drawn on admission for the following measurements: (1) bloodglucose and urea nitrogen, sodium, potassium, chloride, total calcium,phosphate, magnesium, creatinine, uric acid, total cholesterol,triglycerides, LDL-cholesterol, HDL-cholesterol, serum aspartate andalanine aminotransferases, gamma-glutamyl transpeptidase,5'-nucleotidase, alkaline phosphatase, serum protein electrophoresis,complete blood count, prothrombin time, activated partial thromboplastintime, fibrinogen and alpha-fetoprotein; (2) serum lgG, lgA and lgM,determined by radial immunodiffusion (Behring Diagnostics, Madrid); (3)serum C4, determined by hemolytic titration (Gaither et al, J. Immunol.113:574 (1974)), and serum C3 and C3a desArg, determined by radialimmunodiffusion (Behring Diagnostics); (4) plasma levels of zinc,measured by absorption spectrophotometry (pye Unicam SP 190); (5)circulating immune complexes, determined by [¹² I]Clq binding (Zublerand Lamber In: Bloom and David, eds. In vitro Methods in Cell-Mediatedand Tumor Immunity, New York: Academic Press pp 565-72(1976)); (6)peripheral-smear examination after Wright-Giemsa staining to assess thepresence of Howell-Jolly bodies as an index of splenic function (Boykoet al, Am. J. Clin. Pathol. 77:745 (1982)) (negative in all patients);(7) macrophage Fcγ-receptor-dependent clearance in vivo; and (8)Fcγ-receptor-mediated recognition of sensitized cells byperipheral-blood monocytes in vitro; and (9) abdominal ultrasound toassess for the presence of splenomegaly, which was detected in 17 out ofthe 49 patients.

Preparation of human IgG anti-Rh(D):

Human IgG anti-RH(D) was prepared from serum from a single donor (wasHIV-1 negative by ELISA-Pasteur Institute, Madrid-, WesternBlott-Pasteur Institute, Madrid- and the quantitative end-point dilutionmethod) by ammonium sulfate preciptation followed by Sephacryl S-300 gelfiltration and QAE ion-exchange chromatography (Pharmacia, Madrid). NoIgM was detected by double immunodiffusion (Ouchterlony analysis). Thefinal IgG fraction was passed through a Millipore filter and tested forpyrogenicity and sterility. The final IgG fraction was HIV-1 negative byELISA (Pasteur Institute, Madrid), Western Blott (Pasteur Institute,Madrid) and the quantitative end-point dilution method (Ho et al, N.Engl. J. Med. 321:1621 (1989)).

Macrophage Fcγ-receptor-mediated clearance:

Clearance studies were performed as previously described (Ruiz et al, N.Eng. J. Med. 322:717 (1990); Frank et al, N. Engl. J. Med. 300:518(1979); Schreiber and Frank, J. Clin. Invest. 51:575 (1972)). In brief,erythrocytes (RhD) were isolated from all subjects, washed three timesin physiologic saline, spectrophotometrically standardized to aconcentration of 6.6×10 cells per milliliter, and radiolabeled with ⁵¹Cr (potassium dichromate, Amersham, Buckinghamshire, England). Analiquot of cells was sensitized by adding to it drop by drop anappropriate dilution of the purified human IgG anti-Rh(D). The mixturewas incubated at 37° C. for 30 minutes, and the sensitized ⁵¹ Cr-labelederythrocytes were washed four times in saline and resuspended to aconcentration of 3.3×10 per milliliter in Hanks' balanced salt solution(M.A. Bioproducts, Madrid). An aliquot of cells (usually 10 ml, with 2.5μCi of radioactivity) was injected through an antecubital vein, and thesurvival of red cells was determined in serial blood samples obtainedover a period of 48 hours. Clearance curves were plotted by expressingthe number of counts per minute at each time point as a percentage ofthe number of counts at 10 minutes, the zero point. The time requiredfor clearance of the 50 percent of the inoculated IgG-coated red cells(half-time) was calculated and then correlated with clinical andserologic data. In addition, for the clearance on each day, thepercentage for the inhibition of clearance above control was calculatedat 1, 1.5, 2, 8, 24 and 48 hours, according to the formula ##EQU1##where CPMb denotes the number of counts per minute in a control subjectwho received an injection of unsensitized autologous red cells, CPMx thenumber of counts in a patient who received IgG-coated (sensitized)autologous red cells, and CPMc the number of counts in a control subjectwho received autologous IgG-sensitized red cells. By means of thisformula, patients could be compared with controls studied on the sameday, and results could be expressed as the percentage of change inclearance, where 100 percent inhibition of clearance indicated thatclearance in a patient who received IgG-coated red cells (CPMx) wasidentical to clearance in a control who received unsensitized red cells(CPMb) (Friedman, J. Clin. Invest. 75:162 (1985)). In three additionalcontrol groups--five patients with alcoholic cirrhosis of the liver,five non-cirrhotic alcoholic subjects, and five healthy volunteers--theclearance of autologous ⁵¹ Cr-labeled but unsensitized red cells and theclearance of ⁵¹ Cr-labeled heat-damaged autologous red cells wereexamined.

Duplicate studes were performed in nine of the patients with alcoholiccirrhosis of the liver in whom severe infection had developed, six ofthe patients with alcoholic cirrhosis of the liver without a history ofcomplications due to infection, and six controls. The results of therepeat studies of clearance were unchanged from those of the originalstudies in each subject. Serum C3, C3a desArg, and C4 were measured toassess complement activation during the clearance of IgG-coated redcells. No significant complement activation was observed in any of thepatients included in the present study.

Number of IgG (Anti-RhD) molecules per red cell:

The number of IgG molecules per red cell was determined as previouslydescribed with the use of ¹² I-labeled anti-IgG reagent (Cines andSchreiber, N. Engl. J. Med. 300:106 (1979)). Clearance studies werealways performed with erythrocytes sensitized so that approximately 600molecules of IgG were present on each red cell. WhenFcγ-receptor-dependent recognition by blood monocytes was studied invitro, each red cell (RhD) was coated with 400, 800, or 1600 moleculesof lgG.

Binding of IgG(Anti-RhD)-coated red cells:

The recognition of lgG-coated red cells (RhD) by monocytes isolated wasdetermined as previously described (Gomez et al, J. Reticuloendothel.Soc. 31:24 (1982); Schreiber et al, J. Clin. Invest. 56:1189 (1975)). Inbrief, confluent monolayers of 5.5×10 monocytes were obtained fromdefibrinated blood after density-gradient centrifugation(Ficoll-Isopaque) and plastic adherence to petri dishes (Nunc,Amsterdam). An aliquot of 2×10⁷ 51 Cr-labeled, IgG-coated red cells(RhD) was added to each monocyte monolayer. The petri dishes were thenincubated at 37° C. in an atmosphere of 5 percent carbon dioxide for 45minutes, washed to detach unbound red cells, and treated with 0.086MEDTA solution to remove adherent monocytes and monocyte-bound IgG(Anti-RhD)- sensitized red cells. The treatment with EDTA removed alladherent monocytes and all radioactivity. The percentage of ⁵¹Cr-labeled and IgG-sensitied red cells (RhD) recognized byperipheral-blood monocytes was determined according to the formula:##EQU2## No phagocytosis of anti-RhD-sensitized erythrocytes byperipheral blood monocytes occurs under the experimental conditions(Gomez et al, J. Reticuloendothel. Soc. 31:241 (1982); Schreiber et al,J. Clin. Invest. 56:1189 (1975)). The studies were repeated in 6controls, 6 non-cirrhotic alcoholic patients, 9 of the patients in whomsevere infection developed and 6 of the patients with no history ofinfectious complications; the results of the repeat studies wereunchanged from those of the original studies in each subject.

Preparation of IgG2b-sensitized red cells:

Antibody-sensitized sheep erythrocytes (EA) were prepared as previouslydescribed (Rossman et al, Exper. Hematol. 21:177 (1993)). In brief, 1×10sheep red blood cells in 1.0 ml of 0.01 mol/L EDTA buffer weresensitized by adding mouse monoclonal antibody Sp2/HL, subclass IgG2b(Serotec Ltd., Bicester, Oxon), in 0.1 ml at 37° C. for 1 hour. Thefinal antibody dilutions used to prepare these cells were between 1:10and 1:80. The IgG-sensitized (coated) sheep red cells were washed twiceand resuspended in HBSS to a final concentration of 1×10 cells/ml. Inaddition, a polyclonal 7S IgG rabbit anti-sheep red blood cell (CordisLaboratories) was also used to prepare polyclonal IgG-coated red bloodcells. The final antibody dilution used to prepare these cells was1:1000.

Monocyte recognition of sheep IgG-sensitized red blood cells:

Monocyte in vitro recognition of IgG-sensitized red cells was assessedas previously reported (Rossman et al, Exper. Hematol. 21:177 (1993);Schreiber et al, N. Engl. J. Med. 316:503 (1987)). In brief, 1×10IgG-coated red cells or control unsensitized red cells were added tomonocyte monolayers containing 1×10 cells. These cells were incubated at4° C. or 37° C. in phosphate buffer at an ionic strength of μ=0.07 orμ=0.15, respectively. After 2 hours, the plates were washed and stainedwith Wright's Giemsa. Two hundred (200) monocytes were counted underlight microscopy in a blinded fashion to assess the number ofIgG-sensitized red blood cells bound per monocyte. Monocytes binding >3red blood cells/monocyte were determined. These experiments wereperformed in 5 patients of each alcoholic cirrhosis of the liver groups(I, II and, III), 5 alcoholic non-cirrhotic subjects and 5 normalvolunteers. The experiments were repeated in these same patients andcontrols at least one year after the initial studies. No significantvariations were found between the initial experiments and the onesperformed after more than one year.

HLA typing:

HLA typing was performed by the tissue-typing laboratory of the Virgendel Rocio University Hospital, Seville, Spain.

Assessment of nutritional status:

Nutritional status was evaluated according to anthropometric,biochemical, and immunologic measurements (Blumonkrantz et al, Am. J.Clin. Nutr. 33:1567 (1989); Harvey et al, Am. J. Clin. Nutr. 33:1587(1989); Feliffe, Wo 1966, No. 53, Geneva, Switzerland; Bristian et al,JAMA 235:1567 (1976)). Dry body weight, relative body weight, and thepercent ideal body weight were also determined. The anthropometric datawere compared with standard values for the local population (Jaurrieta,Med. Clin. 81:584 (1983)). Serum albumin and transferrin were measuredto evaluate the serum protein level. Malnutrition was classifiedaccording to previously established criteria (Blumenkrantz et al, Am. J.Clin. Nutr. 33:1567 (1980); Harvey et al, Am. J. Clin. Nutr. 33:1586(1980); Feliffe, WO 1966 No. 53, Geneva, Switzerland; Bristian et al,JAMA 235:1567 (1976); Jaurrieta, Med. Clin. 81:584 (1983); O'Keefe etal, Lancet 2:615 (1980)) as marasmus, kwashiorkor, or mixed type. Allmalnourished patients had malnutrition of the mixed type. A highincidence of protein-calorie malnutrition of the mixed type was observedin 17 of the 49 patients (35 percent). Total body weight did not change.Cutaneous hypersensitivity responses to standard concentrations of fourantigens-purified protein derivative, Trycophyton rubrum, Candidaalbicans, and streptokinase-streptodornase-were used to evaluatecell-mediated immunity as previously described (Harvey et al, Am. J.Clin. Nutr. 33:1586 (1980); Blackburn et al, J. Parenter. Enteral. Nutr.1:11 (1977)). A response was considered positive when the diameter ofinduration was more than 5 mm. A normal response was indicated by apositive response to either three or four antigens, an abnormally lowresponse by a positive response to either one or two antigens, andanergy by a lack of positive response to any of the four antigens.

Statistical analyses:

The in vivo clearance curves were analyzed at the time points tocalculate a P value for the difference between the controls and patientsby Student's t-test. The in vitro Fcγ-receptor-dependent recognition ofred cells by monocytes and the clearances in patients and controls wereassessed with the Wilcoxon rank-sum test for unpaired data. The relationof the clearance rate (as half-time) or monocyte Fcγ-receptor-dependentrecognition of IgG-coated red cells in vitro to the seologic tests wasanalyzed with the Spearman rank-correlation test.

Clearance Study Results:

Clearance studies were performed in the 49 patients with alcoholiccirrhosis of the liver fulfilling the inclusion criteria of this study.The results demonstrated that the clearance of IgG-coated red cells wassignificantly impaired (p<0.001) (FIG. 4). At 1 and 1.5 hours, the mean(±SEM) inhibition of macrophage Fcγ-receptor-mediated clearance was 47±3and, 53±3 percent, respectively. Clearance was inhibited by more than 15percent in 37 patients and, by 5 to 12 percent in 6. In contrast, theclearance of unsensitized red cells and of heat-damaged red cells in thepatients did not differ from the clearance of these cells in thenon-cirrhotic alcoholics and healthy volunteers (FIG. 4).

Patients were classified in three groups according to their degree ofliver insufficiency as assessed by the Orrego index. Clearance studiesof those three groups of patients are represented in FIG. 5. The resultsdemonstrated that the clearance of IgG-coated red cells wassignificantly impaired (p<0.001) in patients with moderate (Patients IIor group II) and severe (Patients III or group III) liver insufficiency.At 1 and 1.5 hours, the mean (±SEM) inhibition of macrophageFcγ-receptor-mediated clearance was 47±3 percent and 66±4 percent,respectively, for group II patients. At 1 and 1.5 hours the mean (±SEM)inhibition of macrophage Fcγ-receptor-mediated clearance of lgG-coatedred cells was impaired in patients with mild liver insufficiency(Patients I or group I), (FIG. 5), but the difference was notsignificant.

The patients were followed up for at least two years after the clearancestudies were initially performed. Six patients died, two of massivehemorrhage from ruptured esophgeal/gastric varices (15th and 17th monthof follow up, respectively), two spontaneous bacterial peritonitis by E.coli (14th and 20th month of follow up, respectively), and twoGram-negative sepsis due to E. coli and (16th and 21st month of followup, respectively). Eleven patients had severe infection: five hadspontaneous bacterial peritonitis (E. coli) and, six had sepsis (due toE. coli in three, Staphyloccus aureus in one, in one, and Serratiamarcescens in one). When the clearance of IgG-coated red cells in thepatients with severe infection was compared with the clearance in thepatients without infection, those with infection were found to have asignificantly longer half-time (126.2±22 vs. 32.2±18 hours;p<0.001)(FIG. 6). The clearance of IgG-coated red cells was analyzed inthe patients (half-time) in relation to various parameters of liverimpairment (SGOT, SGPT, GGT, 5'-nucleotidase, bilirubin -total, directand indirect-, P.T., aPTT, fibrinogen and serum albumin). None of theseparameters, including the presence of splenomegaly, correlated with theextent of impairment of clearance of lgG-coated red cells.

Isolated peripheral blood monocytes were also studied (FIG. 7).Erythrocytes from a single Rh(D)-positive donor were sensitized withthree different concentrations of IgG-antiRh(D) (400, 800, and 1600 IgGmolecules per red cell). Monocytes isolated from the patients boundfewer IgG-coated red cells than did those from the controls, but thedifference was not significant. There was no correlation between theextent of binding by monocytes and the degree of impairment of clearanceof IgG-coated red cells. No difference was observed between thisalteration in monocyte FcγRI in patients in whom severe infectiondeveloped and those in whom it did not.

The function of monocyte FcγRII was assessed in vitro by the binding ofIgG2b-coated red blood cells (FIG. 8). Peripheral blood monocytesisolated from patients with cirrhosis of the liver bound lessIgG2b-sensitized red cells than monocytes from non-cirrhotic alcoholicsubjects or monocytes from normal volunteers, but the difference was notsignificant.

Seven patients had elevated levels of circulating immune complexes. Theclearance of IgG-coated red cells in these patients did not differ fromthat observed in the patients in general (FIG. 9). Furthermore, therewas no correlation in these five patients between the level ofcirculating immune complexes and the extent of impairment of therecognition of IgG-coated red cells by monocytes.

Neither the clearance of IgG-sensitized erythrocytes, nor therecognition in vitro of IgG-coated red cells or IgG2b-coated red cellsby monocytes from the patients correlated with their sex, age, time fromdiagnosis of alcoholic cirrhosis of the liver or with any of theserologic measurements, including the immunoglobulin level. Furthermore,there was no relation between either the clearance of IgG-coated redcells or their recognition in vitro by monocytes and the HLA haplotype,or the nutritional status of the population studied.

The plasma zinc level was 18.4±0.7 μmol per liter (120 μg per deciliter)in healthy volunteers and 12.7±1.3 μmol per liter (83.3±3.7 μg perdeciliter) in the patients with alcoholic cirrhosis of the liver(p<0.001). However, there was no correlation between the plasma zinclevel and the degree of impairment of clearance in vivo or the monocyterecognition of IgG-coated red cells in vitro. Similarly, malnutritionwas not necessarily linked with greater impairment of the clearance rateor a lower value for in vitro monocyte recognition of IgG-sensitized redcells. The prevalence of malnutrition was significantly higher in thepatients with either moderate or severe liver insufficiency (groups IIand II, respectively) (p<0.001). However, neither the macrophageFcγ-receptor-mediated clearance nor the binding of IgG (Anti-RhD)-coatedred cells or the binding of IgG2b-coated red cells by monocytescorrelated with the nutritional status of these patients, as indicatedby anthropometric, biochemical, and immunologic values.

EXAMPLE X T-Cells Transfected with FcγRIIA

Experimental Protocols:

Cell lines and antibodies:

The Jurkat T-cell line J32 and the CD2-CD28-CD3+ variant J32-3.2 havebeen described previously (Makni et al, J. Immunol. 146:2522 (1991) andSancho et al, J. Immunol. 150:3230 (1993)). These cell lines weremaintained in RPMI 1640 containing 10% heat inactivated FCS (HycloneLaboratories, UT), 2 mM L-glutamine, penicillin (100 U/ml) andstreptomycin (100 U/ml). The following antibodies were used in thisstudy: anti-CD2 mAbs 9.6 (Sancho et al, J. Immunol. 150:3230 (1993))and9.1 (Yang et al J. Immunol. 137:1097 (1986)), anti-CD3 mAb 64.1 (Hansenet al, In Leukocyte Typing, Bernard et al eds. Springer-Verlag, New Yorkp. 195 (1984)) and anti-FcγRII mAb IV.3 (Fanger et al, Immunol. Today10:92 (1989)).

Construction of the FcγRIIA expression vector and DNA transfer into J32and J32-3.2 cell lines:

FcγRIIA cDNA was isolated from the plasmid pKC4 (Hibbs et al, Proc.Natl. Acad. Sci. USA 5:2240 (1988)) using EcoR1 and the fragment wasblunt ended using Klenow polymerase. The FcγRIIA cDNA was then insertedinto the Smal site of plasmid pGSE1731 (Greaves et al, Cell 56:979(1989)) under control of the human β-globin gene promoter and enhancersequences. pGSE1731 contains 4.9 Kb of the human β-globin gene including1.5 Kb of sequences upstream of the CAP site and the internal and 3'enhancer regions. This plasmid also contains the CD2 3' enhancer regionwhich confers T-cell specific, position-independent gene expression(Greaves et al, Cell 56:979 (1989)). The resulting plasmid, pGSE2A wasintroduced into the J32 and J32-3.2 cell lines by electroporation usingmethods previously described in detail (Sancho et al J. Immunol.150:3230 (1993)). Prior to electroporation, pGSE2A was linearized bydigestion with Notl. Each electroporation was carried out using 30 μg oflinearized pGSE2A and 5 μg of pcEXV Neo linearized with EcoRI. Afterelectroporation, the cells were cultured for seven days in the presenceof 0.3 mg/ml G418 and assayed for FcγRIIA expression by flow cytometry.FcγRIIA expressing cells were enriched by immunomagnetic positiveselection using magnetic particles coated with IgG (Dynal Inc., FortLee, N.J.). Cells were cultured in flat buttomed microtitre wells(approx. 100 cells per well) and clones were selected and analyzed forFcγRIIA expression by flow cytometry.

Tyrosine Phosphorylation Results:

Stimulation of the T-cell receptor (TCR)/CD3 complex in Jurkat T-cellsinduces the tyrosine phosphorylation of proteins including theTCR-associated ζ chain, the ZAP70 tyrosine kinase and the CD3ε complex(Weiss, Cell 79:209 (1993)). Similarly, in the members of the IgG familyof receptors, induction of tyrosine phosphorylation accompanies receptoractivation (Samelson and Klausner, J. Biol. Chem. 267:24913 (1992)) and,accordingly, studies were conducted to determine if stimulation ofFcγRIIA in the T-cell transfectants J32/FcγRIIA and J32-3.2/FcγRIIAinduced tyrosine phosphorylation. The mutant J32-3.2 cell line isdeficient in the induction of tyrosine phosphorylation signallingpathways leading to impaired induction of phosphorylated ZAP70, ζ chainand CD3ε after TCR crosslinking (Sancho et al, J. Immunol. 150:3230(1993)). The activation of the Src-related tyrosine kinase (SRTKs)p561ck and p59fyn is also defective in this mutant (Sancho et al, J.Immunol. 150:3230 (1993)).

Stimulation of FcγRIIA by crosslinking with anti-FcγRII antibodyfollowed by immunoprecipitation with anti-phosphotyrosine antibody(Huange et al, J. Biol. Chem. 267:5467 (1992)), showed that the 40 kDFcγRII receptor is phosphorylated on tyrosines in both wild-type J32 andin the mutant J32-3.2 transfectants (FIG. 10, lanes 4-11 (the positionof the 40 kD receptor is indicated with an arrow).

Phagocytosis Results:

FcγRIIA cDNA was expressed in the wild type Jurkat T-cell line J32 andin the mutagenized J32 variant, J32-3.2. As noted above, the J32-3.2cell line is CD2-CD28-CD3+ and exhibits reduced signal transductioncapabilities after TCR/CD3 stimulation, with respect to tyrosinephosphorylation pathways and GTP binding mechanism (Sancho et al, J.Immunol. 150:3230 (1993)). Calcium mobilization and IL2 promoteractivity induced after TCR stimulation are also impaired (Sancho et al,J. Immunol. 150:3230 (1993)). Fluorescence histograms of J32/FcγRIIA andJ32-3.2/FcγRIIA stable transfectants, and FcγRIIA expressing clonesisolated from these transfected cells, are shown in FIG. 11.

The ability of these T-cell transfectants to phagocytose IgG-sensitizedcells was assessed by incubation with IgG coated sheep erythrocytes(sEA). In both the wild type J32 and mutant J32-3.2 transfectants, anumber of the cells were able to phagocytose the sEA (FIG. 12). Theresults of several experiments with (a) bulk cell stableFcγRIIA-transfectants and (b) FcγRIIA clones are shown in Table 1. Thedata indicate that these T-cell transfectants phagocytose EA and thatphagocytosis by the J32-3.2 mutant transfectants was reduced compared tothe wild type cells.

                  TABLE 1                                                         ______________________________________                                        Phacocytosis of Sheep EA by FcγRIIA bulk cell stable                    transfectants of J32 and J32-3.2 cell lines.                                  J32/FcγRIIA                                                                             J32-3.2/FcγRIIA                                         %            P1     %          P1   cP1                                       ______________________________________                                        1.     17        28     --       --   --                                      2.     33        22      5        6    7                                      3.     25        40     13       18   21                                      4.     32        53     17       21   24                                      5.     31        51     --       --   --                                      6.     14        21      6        8    9                                      ______________________________________                                    

P1 is the phagocytic index, i.e., the number of erythrocytes ingestedper 100 cells. The corrected P1 value (eR1) is included in theJ32-3.2/FcγRIIA column to take into account the lower MF1 value observedin these transfected cells compared to the J32/FcγRIIA transfectedcells. %=% phagocytic cells.

Considering that 70%-100% of the cells are expressing FcγRIIA in thesetransfectants, and presumably are mediating phagocytosis through thisreceptor, the levels of phagocytosis observed are relatively low whencompared, for example, to COS-1 fibroblasts transfected with FcγRIIA(Indik et al, J. Clin. Invest. 88:1766 (1991)). However, the ingestionof the erythrocytes appears to be mediated via a genuine phagocyticprocess as preincubation of the cells in 10 μg/ml cytochalasin-D, acompound which inhibits actin polymerization (a process that isnecessary for phagocytosis) (Indik et al, J. Clin. Invest. 88:1766(1991)), abolished phagocytosis in these cells. Also phagocytosis wasinhibited when the transfectants were incubated with sEA at 0° C.instead of 37° C.

All documents cited hereinabove are incorporated in their entirety byreference.

While the invention has been described with respect to what is presentlyregarded as the most practical embodiments thereof, it will beunderstood by those of ordinary skill in the art that variousalterations and modifications may be made which nevertheless remainwithin the scope of the invention as defined by the claims which follow.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 7                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ACGATGTCTAGAGGTGACTTGTCCACTCC29                                               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CAGGATATAGCAGAGTTGGTACCCAGGTGG30                                              (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CCACCTGGGTACCAACTCTGCTATATCCTG30                                              (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       ATGGCGAGCTCTCCGGTAAACAGCATCTGAG31                                             (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GluXaaXaaXaaXaaXaaXaaXaaXaaAspXaaXaaTyrXaaXaa                                 151015                                                                        LeuXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaTyrXaaXaaLeu                           202530                                                                        (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /label=Xaa                                             /note="first and third Xaa is D/E; second Xaa is                              (Xaa)2,7; fourth and sixth Xaa is (Xaa)2; and                                 fifth Xaa is (Xaa)7"                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       XaaXaaXaaTyrXaaLeuXaaTyrXaaLeu                                                1510                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       TyrXaaXaaLeu                                                                  __________________________________________________________________________

What is claimed is:
 1. A DNA construct encoding an Fcγ receptorcomprising extracellular, transmembrane and cytoplasmic domains, orfunctional portions thereof, from at least two of human FcγRI, humanFcγRIIA, the α chain of human FcγRIIIA, and the γ chain of humanFcγRIIIA,wherein the transmembrane domain of said receptor is not thatof the α chain of human FcγRIIIA and wherein said domains, or portionsthereof, are such that said Fcγ receptor renders phagocytic a COS-1 cellcomprising same.
 2. The construct according to claim 1 wherein saidcytoplasmic domain is the cytoplasmic domain of FcγRIIA or of the γchain of FcγRIIIA.
 3. The construct according to claim 1 wherein saidreceptor comprises the extracellular domain of the α chain of FcγRIIIA,the transmembrane domain of the γ chain of FcγRIIIA or FcγRI, and thecytoplasmic domain of the γ chain of FcγRIIIA.
 4. The constructaccording to claim 1 wherein said receptor comprises the extracellulardomain of FcγRIIA and the transmembrane and cytoplasmic domains of the γchain of FcγRIIIA.
 5. The construct according to claim 1 wherein saidreceptor comprises, in a single cytoplasmic domain thereof, at least twocopies of the sequence Y-X2-L, wherein X2 represents any two aminoacids.
 6. The construct according to claim 5 wherein X2 represents theamino acids of a Y-X2-L sequence of the cytoplasmic domain of FcγRIIA orthe γ chain of FcγRIIIA.
 7. A DNA construct encoding an Fcγ receptorcomprising, in a single cytoplasmic domain thereof, at least one copy ofthe sequence Y-X2-L in addition to the corresponding naturally occurringcytoplasmic domain, wherein said naturally occurring cytoplasmic domainincludes at least one Y-X2-L sequence, wherein X2 represents any twoamino acids and wherein said Fcγ receptor renders phagocytic a COS-1cell comprising same.
 8. The construct according to claim 7 wherein X2of said at least one additional Y-X2-L sequence represents the aminoacids of a Y-X2-L sequence of the cytoplasmic domain of FcγRIIA or the γchain of FcγRIIIA.
 9. The construct according to claim 7 wherein onecopy of the Y-X2-L sequence is present in addition to the correspondingnaturally occurring cytoplasmic domain.
 10. A DNA construct encoding anFcγ receptor comprising, in a single cytoplasmic domain thereof, atleast one duplication in the number of Y-X2-L sequences present in thecorresponding naturally occurring cytoplasmic domain, wherein saidnaturally occurring cytoplasmic domain includes at least one Y-X2-Lsequence and wherein X2 represents any two amino acids and wherein saidFcγ receptor renders phagocytic a COS-1 cell comprising same.
 11. Theconstruct according to claim 10 wherein X2 represents the amino acids ofa Y-X2-L sequence of the cytoplasmic domain of FcγRIIA or the γ chain ofFcγRIIIA.
 12. The receptor according to claim 10 wherein said naturallyoccurring cytoplasmic domain is the cytoplasmic domain of FcγRIIA, the γchain of FcγRIIIA or of FcεRI, and wherein X2 represent the amino acidsof the Y-X2-L sequence of the cytoplasmic domain of FcγRIIA or the γchain of FcγRIIIA or FcεRI.
 13. A cell comprising the constructaccording to one of claims 1, 7 and
 10. 14. The cell according to claim13 wherein said cell is non-phagocytic prior to introduction of saidconstruct.
 15. The cell according to claim 14 wherein said cell is afibroblast.
 16. The cell according to claim 13 wherein said cell is anepithelial cell.